This invention relates to flow cytometers as are used in clinical and research applications. Specifically, the invention relates to systems which monitor the conditions of the jet emitted from a nozzle in a flow cytometer during the processing operation. The invention may be applied regardless of the type of processing involved and is particularly appropriate for sorting and analysis applications.
Flow cytometers have been in clinical and research use for many years. Basically, the systems act to position small amounts of a substance within a sheath fluid. This sheath fluid may either form droplets or may exist in a jet for optical analysis. Through hydrodynamic focusing and laminar flow, the substance is split into individual cells and the like and is surrounded by a sheath fluid. In many applications, the sheath fluid together with its entrained substance exits a nozzle in a jet and either free falls or is channeled in an optically transparent pathway for analysis. This analysis requires very precise and uniform conditions within the jet. Unfortunately, variations do still exist. Thus, there has been a need to monitor the conditions of the jet in order to assure accurate analysis. Two types of variations are of particular concern. First, the breakoff point at which droplet formation occurs tends to vary or drift. This exists as a result of changes in material, temperatures, the existence of air bubbles, and the like as those skilled in the art well know. Since the exact location of the droplet separation point is utilized for differentially charging various droplets and other aspects, such variations can destroy the ability to achieve the desired function of the flow cytometer.
The change in droplet separation point has been the subject of a number of inventions. As U.S. Pat. Nos. 4317520, 4318480, 4,318,481, 4,318,482, 4,318,483, and 4,325,483 explain, traditionally the approach has been to determine the droplet separation point through the use of a strobe light and microscopic analysis prior to the actual processing of the substance. The processing is then conducted based upon the location determined for the droplet separation point under the assumption that no drift or variation occurs. Obviously, this type of approach does not recognize the realities of actual processing and totally ignores the fact that variation in the location of the droplet separation point does, in fact, occur.
As U.S. Pat. Nos. 4,317,520, 4,318,480, 4,318,481, 4,318,482, 4,318,483, and 4,325,483 further explain, one approach taken has been to utilize the amount of scatter of the light emitted from a laser fluorescent source as an indirect indication of the location at which the surface tension "pinching" (and thus the droplet formation) is occurring. This approach has the significant advantage of affording the opportunity to actually monitor the conditions during processing, however, it does not provide a direct indication of the condition. While a great improvement over the traditional approach, the approach these related patents take still appears to provide only an indirect--and somewhat less accurate--indication of the exact location of the droplet separation point. Further, these references also suggest illumination of the sheath fluid at a wavelength where the sheath fluid is translucent. This can be unnecessary.
Similarly, related U.S. Pat. Nos. 4,691,829 and 4,487,320 suggest the use of scattered light as an indirect indication of the droplet separation point. These two references each explain that while a direct indication of the droplet separation point would be desirable, a physical limitation makes this impractical. Basically, they explain that the presence of undesired light destroys the ability to gather the data desired, namely, the emissions from the substance itself.
A second type of variation in the conditions within the jet which is of concern is the fact that the jet can tend to move horizontally. This is true even for systems in which the jet is channeled. While, naturally, the nozzle container is usually fixed, the same type of variations which cause change in the droplet separation point can also cause the jet to be directed to one side or the other. While this amount of variation can be relatively small, it still poses an unnecessary degradation in the signal sensed as it can change the focal point and otherwise destroy resolution accuracies. None of the references mentioned seem to have addressed this aspect.
One of the practical problems which has also been recognized is the fact that only a limited amount of space exits within which to conduct monitoring and sensing. As Japanese Patent 2024535 recognizes with respect to the sensing system alone, it is desirable to have an optical system which is as small as possible. The present invention achieves this as well as other goals.
As mentioned, there has been a long felt but unsatisfied need for a device which permits accurate and direct monitoring of the conditions within the jet of a flow cytometer during the processing operation. As the present invention shows, such a system is, indeed, possible and, in fact, can be implemented using arts and elements which had long been available. To some extent, apparently solutions had not been achieved because those skilled in the art seem to have taken a direction which was away from the technical direction pursued in the present invention. This may have been the result of the fact that those skilled in the art did not truly appreciate the nature of the problem or may have been the result of the fact that those skilled in the art were misled by some of the presumptions and assumptions with respect to the type of systems which could be considered. In this regard, it had been assumed that direct monitoring through the use of some illumination source or some optical source was not practical because of an inevitable interference with the sensing system. The present invention shows that this is not the case and that direct monitoring can be achieved without the interference. Not only in this regard but in other facets as well, the present invention demonstrates that although substantial attempts had been made by those skilled in the art in order to achieve a practical monitoring system, the direction of the present invention was not recognized. Until the present invention, a system which allowed direct monitoring of jet conditions was not practically achievable.